1. PARP Family and Structural Characteristics
It has been more than 50 years since the poly(ADP-ribose) polymerase (PARP) was firstly observed by Chambon and coworkers thereof in 1963, and PARP has attracted the attention of many researchers because of the applications in the repair of damage and maintenance of genome stability. Out of PARP family of enzymes, PARP-1 is the first found and extensively studied enzyme with the most typical structure. PARP-1 plays a key role in DNA repair, apoptosis, proliferation, etc., which is regarded as “the guardian angel of DNA”. The poly(ADP-ribose) polymerase (PARP) is present in eukaryotes, catalyzes nicotinamide adenine dinucleotide (NAD+) to release ADP-ribose, and further catalyze ADP-ribose to polymerize at the specific sites of various important proteins including PARP itself to form polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR), thereby regulating the function of the protein and playing key roles during the repair of the single-strand DNA breaks.
PARP constitutes a family of cellular ribozyme proteins that catalyze the synthesis of poly(ADP-ribose). So far, 18 members in this family have been isolated and identified including: PARP-1, PARP-2, PARP-3, vPARP (PARP-4), Tankyrase-1 (PARP-5), Tankyrase-2 (PARP-5b), PARP-6, tiPARP (PARP-7), PARP-8, PARP-10, PARP-11, PARP-12, ZAP (PARP-13), BAL-1 (PARP-9), BAL-2 (PARP-14), BAL-3 (PARP-15), PARP-16, PARG. Among them, PARP-1 is the earliest discovered and the most well-known member of the PARP family, and the activity thereof accounts to more than 90% of the total cellular PARP activity. It is a polypeptide chain composed of 1014 amino acids having a molecular weight of 116 kDa. including three main functional domains: N-terminal DNA binding domain (DBD), auto-modification domain (AMD), and C-terminal catalytic domain. The DNA binding domain (DBD) contains two zinc-finger (referred as ZnF1 and ZnF2 below) motifs and a nuclear localization sequence. These two zinc-finger motifs are involved in the recognition of DNA nicks. The ZnF1 recognizes single-strand DNA damage and double-strand DNA damage, and its mutation can significantly reduce the activity of the PARP; the ZnF2 can only participate in the recognition of the single-strand DNA damage. The auto-modification domain of PARP-1 contains 15 glutamate residues as targets for ADP ribosylation itself, which is the main regulatory site. The C-terminal catalytic domain is the basis for the conversion of NAD+ to ADP ribose.
In the PARP family, PARP-1 and PARP-2 share the highest homology (up to 69%). Therefore, all reported PARP-1 inhibitors have considerable inhibitory activity against PARP-2 till now.
2. PARP and Disease Treatment
The DNA damages are repaired mainly via base excision repair (BER) or homologous recombination (HR) repair under normal conditions. PARP and BRCA are the major enzymes involved in base excision repair and homologous recombination repair, respectively. For most of the ovarian cancer and triple negative breast cancer patients, two hypotypes BRCA1 and BRCA2 of BRCA usually have mutation, resulting in the loss of DNA damage repair ability, thus cell repair is mainly performed through base excision repair in which PARP enzymes are involved. Cancers could be treated effectively if the function of PARP enzyme to repair DNA damage is blocked, which results in apoptosis.
According to statistics, the prevalence of BRCA1 mutations was 45% for families with multiple cases of breast cancer, and was 90% for families with high incident breast and ovarian cancers. BRCA1 mutations have also been described in sporadic breast cancer cases. In addition, BRCA1/2 mutations are also found in other solid tumors such as ovarian cancer.
In 2005, Bryant and Framer respectively reported that using PARP inhibitors in cells lacking BRCA1/2-mediated homologous recombination repair function, thereby inhibiting PARP-mediated base excision repair (BER) pathways, ultimately causing synergistic lethality of tumor cells. This indicates that PARP inhibitors may be employed alone for the treatment of certain tumors. The results of this study quickly attracted wide attention from pharmaceutical companies and academia. Therefore, new era was opened for the development of PARP inhibitors as highly selective antitumor drugs. Recently, PARP-1 has been identified as a potential therapeutic target for the study of antitumor drugs.
3. PARP Inhibitors
It has been reported by Armin et. al. that the catalytic active sites of PARP-1 can be roughly divided into two domains, donor domain and acceptor domain, both using PARP substrate NAD+ as a scaffold. Acceptor domain binds to ADP of polymeric adenosine diphosphate ribose chains. Donor domain binds to NAD+, and this site can be further divided into three sub-binding domains: nicotinamide-ribose binding site (NI site), phosphate binding site (PH site), and adenosine-ribose binding site (AD site), respectively. Most of the PARP inhibitors interact with the NI site of PARP and competitively inhibit NAD+. Therefore, their structures are similar to nicotinamide. For example, AZD2281 (olaparib/KU-59436) developed by AstraZeneca is an oral small molecule PARP inhibitor, which has shown promising therapeutical effects in treating ovarian cancer, breast cancer and solid tumor in combination with drugs such as cisplatin, carboplatin, paclitaxel and so on. Currently, AZD2281 is on the market.

However, the compound AZD2281 showed weak selectivity and inhibitory activity against PARP-1. The effective dose of the inhibitory activity at the cellular level was 200 nM, and the in vivo dose above 100 mg just showed significant anti-tumor activity. The clinical daily dose is also up to 400 mg (50 mg capsules, 8 capsules). The in vivo action time and half-life time of compound AZD2281 are relatively short (<1 hours), and its bioavailability is also low (<15%). The metabolites of the compound AZD2281 mainly result from the oxidation and deacylation of piperazinyl moiety in the hydrophilic region of the molecule.
Therefore, the structurally stable piperazinotriazine moiety A was introduced in the previous stage by the inventor's team. With optimization of substituent on the piperazine ring, the introduction of methyl group is found to effectively improve the stability of the piperazine substituent moiety and reduce the production of toxic products (CN 103570725 A). Consequently, the triazine fragment was further optimized in the present invention based on the mode of action of PARP enzyme and small molecule, and the derivative B substituted by aryl, heteroaryl, or heterocycloalkyl was found to have relatively high activity.